Since the identification of the gene responsible for the Wiskott- Aldrich syndrome (WAS), we have focused our research efforts on the WAS protein (WASP). The goal of this proposal is to define the molecular basis for classic WAS and its milder form, X-linked thrombocytopenia (XLT), and to study the many functions attributed to WASP in human and murine systems. Because of their central importance for the function of WASP, we have selected the pleckstrin homology (PH) domain and the SH3 binding domain of WASP for detailed analysis. Mutation analysis of patients with WAS/XLT has identified many missense mutations within the PH domain that result in XLT. PH domains are known to bind to membrane lipids (e.g., PIP2) and thus are responsible for localizing PH domain containing proteins to the cell membrane. Using an in vitro binding system, we will investigate whether naturally occurring mutations within the PH domain interfere with the binding of WASP to PIP2 and if site directed mutagenesis generates PH domains that no longer bind to PIP2. In contrast, mutations within the SH3 binding domain of WASP result in a severe WAS phenotype. Naturally occurring mutations and mutations obtained by site directed mutagenesis of the SH3 binding domain of WASP, expressed as GST-fusion proteins, will be used to demonstrate a loss of binding to SH3 containing adapter proteins and kinases known to interact with normal WASP. The effect of mutations within the PH and SH3 domain on tyrosine phosphorylation of WASP will also be investigated. Based on the observation that lymphocytes from patients with classic WAS, but not with XLT, show accelerated apoptosis and increased caspase-3 activity, we will investigate different death pathways to identify the mechanisms leading to this accelerated apoptosis. Finally, we have established a breeding colony of WAS deficient (KO) mice that will allows us to study in vivo the immune defect caused by mutations of WASP, using a T cell dependent antigen that is given at low or high doses by different routes to determine antibody responses, and an in vivo HSV infection model to study the generation of antigen-specific CTLs. The usefulness of WASP KO mice to study abnormal platelet function and accelerated apoptosis in vivo will be explored. Results from these investigations will clarify the function of WASP, explain the phenotypes of WAS/XLT and will undoubtedly have implications for optimal therapy of affected patients.